The binding of chloride and/or CO(2) to hemoglobin (Hb) lowers the oxygen affinity. Several binding sites are present for chloride, of which only the highest affinity site is not sensitive to oxygen binding. One proposal locates an oxylabile chloride binding site in the vicinity of the four alpha-NH(2)-terminal residues of Hb. These sites are also where the binding of CO(2) as carbamate contributes to CO(2) transport. The CO(2) binding and/or chloride binding to the same allosteric effector sites lower(s) the oxygen affinity of hemoglobin. With differential calorimetry and (13)C NMR, the enthalpy of CO(2) binding to isoionic deoxyHb has been found as a function of chloride concentration. In addition, the enthalpy of chloride binding to isoionic deoxyHb has been examined in order to characterize thealpha-NH(2)-terminal residue binding sites.